Saleh, H., Golian, A., Kermanshahi, H., & Mirakzehi, M. T. (2018).

Antioxidant status and

thigh meat quality of broiler chickens fed diet supplemented with α-tocopherolacetate,
pomegranate pomace and pomegranate pomace extract. Italian Journal of Animal
Science, 17(2), 386-395.

Salah et al. (2018) studied the effects of supplementation of α-tocopherol (200 mg kg −1), PPE
(0.1, 0.2 and 0.3 g kg−1) and PP (1, 2 and 3 g kg−1) in broiler diets. The trial was conducted
for 42 days. Feed efficiency was significantly improved by supplementing chickens fed diet
with 0.2 g kg−1 PPE. Long chain polyunsaturated fatty acids (LC PUFA) n-3 level was higher
in the thigh of broilers fed with α-Toc and PPE diets (except 0.1 g kg −1) than in chickens fed
control and PP diets (p < .05). Total phenolic content, lipid peroxidation level and 1,1-
diphenyl-2-picrylhydrazyl radical scavenging activity in the thigh meat were significantly
improved when chickens were fed diets containing α-Toc and PPE (p < .05). Serum lipid
peroxidation level was reduced in chickens fed diets supplemented with PPE (except 0.1 g
kg−1) and α-Toc. In conclusion, the broiler thigh meat may be successfully enriched with LC
PUFA n-3 and its antioxidant potential and quality characteristics may be improved by
supplementing diets with 0.2 and 0.3 g kg−1 PPE. Moreover, the antioxidant potential of PPE
supplementation was equal to that of α-tocopherol acetate in refrigerated meat. BWG was
lower than control in comparision to 0.1g PPE and 1g PP per kg of diet comparision to
control diet.A significantly higher BWG was reported in broilers fed 2g and 3g PP per kg of
diets. Supplementation of PP in different dosase decreased feed efficiency in broiler
chickens.Similary at 0.1g And 0.2g PPE in broiler diets feed efficiency was not improved.

The inclusion of PP into diets impaired growth performance of chickens. The addition of 15 g
kg−1 PP into diets caused decreased BW and FI In general, relatively high dietary
concentrations of polyphenols caused by the addition of these ingredients, reduced
performance in chickens as well as in other livestock animals (Goñi et al. 2007). in broilers
(Rajani et al. 2011). Nevertheless, in our study, reduced chickens performance was only
observed in birds fed diets with PP. The low concentration of condensed tannins present in
the PPE diet may be relatively low to produce a growth depression.

Saleh et al. (2017) reported that dietary PPE reduced the feed efficiency, improved
the nutritional value of meat, enhanced humoral immune response, decreased
plasma triglycerides concentration, and reduced lipid peroxidation of broiler chickens breast
meat without affecting the liver enzyme biomarkers, cholesterol and low-density lipoprotein
(LDL) concentrations, and cellular immune response. Furthermore, the addition of 20 g
PPP/kg diet enhanced the immune function and proliferation of beneficial bacteria in the ileal
and cecal digesta of broiler chickens, while reducing the abundance of pathogenic ones
(Rezvani et al., 2016). Rajani et al. (2011) reported that incorporation of PPP in broiler
chickens diets did not improve the growth performance of broiler chickens, but reduced the
ascites-related mortality and lipid oxidation of meat under refrigerated storage.

The large amount of phenolics present in PP and PPE diets may cause its strong antioxidant
ability, extend the shelf life and improve the quality of meat products. Sáyago-Ayerdi et al.
(2009) indicated that the inhibition of TBARS values in meat could be due to a protective
effect derived from the polyphenols present in added grape pomace which may act similarly
to vitamin E on the lipid bilayers in the meat. Goñi et al. (2007) demonstrated that grape
pomace concentrate supplementation was equal in antioxidant potential to vitamin E. Based
on observations of Rajani et al. (2011), diet containing 15 g kg−1 pomegranate peel was able
to reduce the TBARS occurrence in meat of birds in comparison to control birds.

In conclusion, the broiler thigh meat may be successfully enriched with long chain
polyunsaturated fatty acids n-3 and its antioxidant potential and functional quality
characteristics may be improved by diet supplementation with 0.2 and 0.3 g kg −1 PPE.
Moreover, the PPE supplementation is as effective as α-tocopherolacetate one in enriching
broiler meat and exerts much more pronounced effect than PP.

Sharifian et al. (2019) evaluated the effects of dietary supplementation of PPE (0, 250, 450,
and 650 mg)/kg of diet on male broiler chickens reared under heat stress for 42 days
experimental period. The broiler chickens were subjected to HS (37 ± 1 °C for 7 h/d and
21 ± 1°C for 17 h/d) from d 25 to 42. Dietary PPE linearly significantly increased the BWG
during overall experimental period. PPE supplementation quadratically decreased the Feed
intake (FI) during the starter period. Furthermore, PPE supplementation linearly improved the
FCR during the finisher and overall experimental periods (42days). Dietary PPE addition did
not affect the relative weights of carcass, breast, thigh, abdominal fat, liver, pancreas,
gallbladder, heart, gizzard, bursa of Fabricius, and spleen.
Dietary supplementation with PPE linearly reduced the plasma triglycerides and cholesterol
The primary total anti- sheep RBC titre linearly increased with dietary PPE addition
Furthermore, dietary PPE linearly increased the concentration of plasma glucose at the
end of the study.

It also linearly enhanced the secondary total (P = 0.024) and Ig G (P = 0.045) anti-SRBC
titers. The dietary treatments did not affect the jejunal VH, villus width, and VH/CD ratio.

It is concluded that dietary supplementation with PPE, at levels up to 650 mg/kg, improved
the growth performance and plasma lipid profile of broiler chickens reared under HS.
Furthermore, dietary PPE addition also preserved the quality of meat during refrigerated
storage.

In a recent study, Ahmed and Yang (2017) reported that dietary supplementation with
pomegranate by-products reduced the FI and FCR of broiler chickens without affecting the
BWG.
The results of the present study confirmed the results of Kishawy et al. (2016), who reported
that feeding rancid oil to broiler chickens caused negative changes in the lipid profiles and
supplementation with PPE completely removed the deteriorative effects of rancid oil and
lowered the concentrations of total serum cholesterol, TG, and LDL.

Moreover, Saleh et al. (2017) demonstrated that both the PPP and PPE decreased the TG
concentration, but did not change the total cholesterol and LDL concentrations in broiler
chickens raised under thermoneutral conditions.
The PPE beneficially influenced the plasma lipid profile possibly via decreased absorption
and increased fecal excretion of cholesterol and decreased the activities of 3-hydroxy-3-
methylglutaryl coenzyme A (HMG-CoA) reductase and sterol O-acyltransferase, 2 key
enzymes for cholesterol metabolism (Esmaillzadeh et al., 2004).
These improvements may be related to an increase in protein digestibility (Oliveira et al.,
2010).
The results of the current study demonstrated that dietary PPE supplementation could
protect the liver from deteriorative effects of HS. The glucose concentration linearly
increased with dietary PPE addition at d 42.
Exposing the broiler chickens to HS reduces the flow of blood and nutrients to the
gastrointestinal tract (GIT), which causes intestinal hypoxia, ATP depletion, intracellular
acidosis, and oxidative and nitrosative stress leading to an alteration in intestinal integrity and
increased membrane permeability to pathogenic microorganisms (Lambert, 2009).
However, dietary PPE addition linearly increased jejunal villus height and linearly tended to
increase the crypt depth and number of goblet cells. The goblet cells of the digestive
tract secrete mucin, which along with other potential functions protect the GIT membrane
from a variety of insults such as pathogenic bacteria and toxic dietary factors (Kindon et al.,
1995).

Broiler chickens under HS may need deeper crypts to provide more functional epithelial cells
to counteract the adverse effects of HS stress (Al-Fataftah and Abdelqader, 2014).

Pomegranate by-products exhibit gastrointestinal protective activity via different

mechanisms.

After ingestion of polyphenol-rich feeds/foods, large amounts of polyphenol metabolites

accumulate in the colon and displays prebiotic properties (Gessner et al., 2017).

For example, Rezvani et al. (2016) reported that feeding of pomegranate peel to broiler
chickens stimulated the growth of Lactobacillus spp., while decreased the abundance of
Escherichia coli in the ileum and cecum.

Pomegranate peel extract as a rich source of antioxidant polyphenols (Ismail et al., 2012),
can protect the GIT against HS-induced oxidative damages.

The authors also found that pomegranate rind extract at the level of 500 mg/kg eliminated
gastric mucosal injury in rats by reducing oxidative stress.

Dietary PPE supplementation did not affect the bursa of Fabricius and spleen relative
weights. However, it linearly increased the primary total anti-SRBC titer. Dietary
supplementation with PPE also linearly enhanced the secondary total and Ig G anti-SRBC
titers.
Furthermore, the primary Ig M response also tended to increase linearly in broiler chickens
fed PPE.

Polyphenols from PPE may improve the immune response of heat-stressed broiler chickens
by improving microbial flora of the intestine (Gessner et al., 2017) and eliminating the free
radicals generated during thermal stress, as well as by regulating the production of cytokines
(Zhao et al., 2016).

Similarly, Saleh et al. (2017) found that PPE supplementation increased the relative weight
of bursa of fabricius and thymus and antibody production against SRBC in the non-stressed
broiler chickens.

However, Prashanth Kumar et al. (2018) reported that dietary PPE did not influence the
antibody titer levels in response to Newcastle disease vaccine and cutaneous basophilic
hypersensitivity in broiler chickens reared under summer conditions.

The results showed that dietary PPE linearly reduced the plasma MDA concentration. The
antioxidant status of muscle is associated with meat quality. Heat stress exposure can
negatively influence muscle antioxidant status and meat quality, causing economic losses.
For example, heat exposure increased serum corticosterone, lightness, drip loss, and muscle
malondialdehyde concentration, while reduced the breast muscle total antioxidant capacity
and catalase and GPx activities in broiler chickens (Zhang et al., 2017).

The lower concentration of MDA in the breast muscles of the PPE-fed chickens suggests that
PPE supplementation can maintain antioxidative status of chicken breast meat as it reduces
lipid peroxidation during meat storage.

Antioxidants, such as polyphenolic compounds, can scavenge ROS within the cell
membranes and therefore, inhibit the initiation or progression of the oxidation process and
thus preserve the meat quality (Goliomytis et al., 2015).

Moreover, iron and copper redox-active forms promote lipid peroxidation; polyphenols are
capable of forming complexes with these metals, thereby protecting muscle cell membrane
from oxidative injury (Goliomytis et al., 2015).
Water holding capacity, defined as the ability of the meat to retain water, affects meat quality
and quantity.

This study also indicated that PPE inclusion in broiler chicken diets also preserved WHC
values of breast meat during storage, which may be associated with improvement in the
antioxidative status of chicken breast meat.

The similar findings were reported by Ahmed et al. (2015) who found that supplemetation
with pomegranate by-products improved the nutritional value and reduced lipid peroxidation
of broiler chicken's meat.

5. Conclusion

Dietary PPE, at levels up to 650 mg/kg, resulted in improvements in growth performance,

plasma lipid profile, antibody responses to SRBC, and intestinal morphology of broiler
chickens reared under HS. Furthermore, PPE supplementation also preserved the quality of
meat during storage. Further studies will be necessary to elucidate the potential of PPE as a
nutritional supplement to alleviate the detrimental effects of heat stress in broiler chickens

Kishawy et al. (2019) studied the effects of addition of 0.5 g and 1 g PPE per kg of basal
diet, on broiler chickens with or without supplementation of soybean oil and linseed oil.
Birds fed diets containing linseed oil without PPE and soybean oil without PPE had higher
(p<0.05) BW and BWG than birds in the other groups, while group fed 0.1 PPE with soy oil
had the lowest BW and BWG than birds in the other groups. The highest FCR (p<0.05) was
recorded for the group fed soybean oil with 0.1% PPE during the starter period. During the
grower period, birds fed soybean oil with 0.1% PPE showed lower BW (p<0.05) than birds in
the other groups. The addition of 0.1% PPE to soybean oil diets resulted in decreased feed
intake (FI) (p<0.05) compared with the other groups; decreased FI also observed in the
linseed oil group without additives and with 0.1% PPE addition. The best FCR was recorded
for group fed soybean oil without additives in grower period. Birds fed 0.1% PPE with
soybean oil or linseed oil had the lowest BW (p<0.05) compared with the other groups; birds
fed linseed oil with 0.1% PPE also had the lower BWG and FI, and the higher FCR, but not
significant with control The highest BW was recorded for groups fed 0.05% PPE with
soybean oil and with linseed oil during the finisher period. In terms of overall performance,
0.05% PPE with soybean oil or linseed oil improved (p<0.05) the BW, BWG, FCR, RGR,
and PER, compared with the values observed with 0.1% PPE with soybean oil or linseed oil.
The PPE supplementation with 0.05% improved final body weight with either soybean oil
ration or linseed oil. The PPE improved carcass dressing percentage in comparison with the
control groups.

Body fat levels decreased with increasing PPE levels, especially with a linseed oil diet.

The PPE supplementation decreased serum total cholesterol levels and increased high-
density lipoprotein cholesterol levels.The content of the breast muscle alpha linolenic acid
improved after replacement of soybean oil with linseed oil in broiler diets. PPE
supplementation increased the phenol and flavonoid content in broiler meat and
increased lysozyme activity.The effects of different dietary levels of PPE with different oil
sources and their interactions on the growth performance of broiler chickens are shown
in Table 4. Birds fed diets containing linseed oil without PPE and soybean oil without PPE
had higher (p<0.05) BW and BWG than birds in the other groups, while group fed 0.1 PPE
with soy oil had the lowest BW and BWG than birds in the other groups.The highest FCR
(p<0.05) was recorded for the group fed soybean oil with 0.1% PPE during the starter
period.During the grower period, birds fed soybean oil with 0.1% PPE showed lower BW
(p<0.05) than birds in the other groups.The addition of 0.1% PPE to soybean oil diets
resulted in decreased feed intake (FI) (p<0.05) compared with the other groups; decreased FI
also observed in the linseed oil group without additives and with 0.1% PPE addition. The best
FCR was recorded for group fed soybean oil without additives in grower period. Birds fed
0.1% PPE with soybean oil or linseed oil had the lowest BW (p<0.05) compared with the
other groups; birds fed linseed oil with 0.1% PPE also had the lower BWG and FI, and the
higher FCR, but not significant with control. The highest BW was recorded for groups fed
0.05% PPE with soybean oil and with linseed oil during the finisher period. In terms of
overall performance, 0.05% PPE with soybean oil or linseed oil improved (p<0.05) the BW,
BWG, FCR, RGR, and PER, compared with the values observed with 0.1% PPE with
soybean oil or linseed oil (Table 5).
Carcass traits

The effects of different dietary levels of PPE with different oil sources and their interactions
on carcass traits of broiler chickens are shown in Table 6. Birds fed PPE at 0.05% and 0.1%
with soybean oil or linseed oil showed significantly higher (p<0.05) carcass dressing
percentages than those in the other groups. The group fed soybean oil without PPE had
significantly higher (p<0.05) intestinal and liver percentage values than the other groups.

Abdominal fat was significantly lower (p<0.05) in groups fed 0.1% PPE with soybean oil
and linseed oil at 0.05% and 0.1%, compared with the other groups.

The highest group in liver and intestinal % was that fed soybean oil without additives.

Lipid profile

The effects of different dietary levels of PPE with different oil sources and their interactions
on the lipid profile of broiler serum are shown in

The addition of PPE to broiler ration, either with soybean oil or with linseed oil, at 0.05% or
0.1% resulted in decreased (p<0.05) total cholesterol, triacylglycerol, LDL cholesterol,
and VLDL cholesterol levels, while the HDL cholesterol concentration increased
(p<0.05) in broiler serum. Generally replacing soybean oil with linseed oil resulted in
decreased (p<0.05) total cholesterol, triacylglycerol, LDL cholesterol, and VLDL cholesterol
levels, while the HDL cholesterol levels increased (p<0.05).

Antioxidant status of broiler breast muscles and bird immunity

The effects of different dietary levels of PPE with different oil sources and their interactions
on the antioxidant status of breast muscles and the immunity of broiler chickens are shown
in Table 9.

The addition of PPE to broiler ration, either with soybean oil or linseed oil, at 0.05% or 0.1%
increased (p<0.05) the total phenol and flavonoid content in muscle; their levels in muscle
increased with increasing levels of PPE in the diet.
Supplementation of soybean oil or linseed oil with 0.1% PPE resulted in improved serum
lysozyme activity and increased the percentage of the bursa of Fabricius and thymus gland to
live BW (Table 6), indicating improved bird immunity.

The results of the current study showed that the addition of 0.05% PPE to poultry diets
improved the final BW, total gain, FCR, RGR, and PER in both soybean oil diets and linseed
oil diets, while with 0.1% PPE supplementation, all of those parameters decreased in
comparison with the values in the control diets.

The interactive effect between linseed oil and PPE in decreasing final BW may be due to
linseed oil had decreased fat precipitation in birds body as reported by [41].

Also the findings from a previous study [42] have shown that rats fed high-fat diets
containing pomegranate leaf extract exhibited decreased obesity through inhibition of
pancreatic lipase, which suppressed lipid digestion and absorption, thus decreasing BW and
weight gain.

Similarly, increased levels of pomegranate peel in rabbit rations have reported to decrease
animal performance and weight gain (Fayed, Azoz, Zedan, and Basyony, 2012).

The results of the present study indicate that birds fed PPE at 0.05% and 0.1% with soybean
oil or linseed oil had higher carcass dressing percentages than those in the other groups; the
group fed soybean oil without PPE had higher intestinal and liver percentages than the other
groups.

Abdominal fat level was lower in the groups fed 0.1% PPE with soybean oil and linseed oil
and in those fed linseed oil with 0.05% PPE compared with the other groups. The current
results are consistent with those reported previously [23,42], which showed that pomegranate
peel or extract or pomegranate leaf extract had an inhibitory effect on lipid metabolism due to
the hypocholesterolemic and hypolipidemic effect in rats fed high-fat diets, thus decreasing
body fat precipitation.

Moreover, the addition of PPE to broiler ration, either with soybean oil or with linseed oil, at
0.05% or 0.1% resulted in decreased total cholesterol, triacylglycerol, LDL cholesterol, and
VLDL cholesterol levels and increased HDL cholesterol levels in broiler serum.
The addition of PPE to linseed oil-containing diets retained the PUFAs of the oil and
improved the precipitation in broiler muscles; this effect may be attributed to the high
antioxidant activity of methanolic extracts of pomegranate peel and their preservative effect
on PUFAs in linseed oil, which prevents their oxidation [24].

The addition of PPE to broiler rations, either with soybean oil or linseed oil, at 0.05% or
0.1% resulted in increased total phenol and flavonoid content in muscles, and their levels in
muscles increased with increasing levels of dietary PPE.

The present study showed that PPE supplementation resulted in a total phenolic content of
600 mg/100 g powder, total flavonoid content of 8.5 mg/100 g powder, and radical-
scavenging activity (I%) of 35.00 at 10 μg/mL.

The high phenol and flavonoid content may be the cause of increased precipitation in broiler
muscles.

Supplementation with 0.1% PPE and soybean oil or linseed oil resulted in improved
lysozyme activity in broiler serum and increased the percentage of bursa of Fabricius and
thymus gland relative to the live BW.

Ross et al. (2001) assessed the effect of Pomegranate fruit rind powder 100 mg/kg provided
orally as an aqueous suspension on cell-mediated and humoral components of the immune
system in rabbits. Rind powder elicited an increase in the antibody titre to typhoid-H antigen.
It also enhanced the inhibition of leukocyte migration in the leukocyte migration inhibition
test, and the duration in a delayed hypersensitivity test with purified protein derivative,
confirming its stimulatory effect on cell-mediated immune response.

Ross, R.G., Selvasubramanian, S. and Jayasundar, S., 2001. Immunomodulatory activity of

Punica granatum in rabbits—a preliminary study. Journal of ethnopharmacology, 78(1),
pp.85-87.

Go to:
CONCLUSION

Addition of 0.05% PPE to soybean oil or linseed oil with the in broiler rations resulted in
improved broiler BW, total BWG, and FCR.

Supplementation of diets with 0.1% PPE decreased serum lipid and abdominal fat levels and
increased flavonoids in broiler muscles.

Hassan et al. (2020) studied the effects of three levels of dietary pomegranate by-product
extract (100, 150, and 200) mg/kg diet on growth performance, nutrient digestibility, carcass
characteristics, and some blood parameters on weaned New Zealand White rabbits at 5 weeks
of age The results revealed that dietary supplementation of pomegranate by-product extract at
each level significantly improved the average final BW and FCR. Rabbits group fed diet
supplemented with 200 mg/kg pomegranate by-product extract recorded the highest of all
nutrients digestibility, DCP, TDN, and DE. Feeding rabbits on diets supplemented with
pomegranate by-product extract at levels 100,150 and 200 mg/kg diet increased plasma
concentrations total protein, albumin, globulin, compared to the control group.

The opposite trend was noticed with glucose, total lipids, triglycerides, total cholesterol
whereas they were lower (P < 0.05) than those of the control group. The obtained results also
showed that pomegranate by-product extract supplementation levels reduced both E. coli and
salmonella counts in rabbit cecum.

It could be concluded that the supplementation of rabbit diets with pomegranate by-product
extract at 100,150 and 200 mg/kg diet improved growth performance and the nutrient
digestibility. Moreover, PBE had an antioxidant and antibacterial effect of the growing
rabbits.

Gungor et al. (2021) revealed that dietary raw pomegranate pomace and fermented
pomegranate pomace (5 and 10 g/kg) did not affect the BW and FCR in broiler chickens.
Supplementation of raw pomegranate pomace and fermented pomegranate pomace (5 and 10
g/kg) improved antioxidant status of the chickens. These results suggest that PP and FP have
the potential to be used in broiler diets as antioxidant and antimicrobial agents. Caecal
Clostridium perfringens count was decreased in broiler chickens of groups 10PP, 5FP and
10FP. However, PP and FP had detrimental effects on the ileum morphology of broiler
chicks. The VH was decreased in the 10PP, 5FP and 10FP groups compared with the control
group. CD was higher in the 5PP and 10FP groups than control and 10PP groups .The
VH/CD ratio was also decreased in 5PP, 5FP, and 10FP groups (p < 0.01).

However, detailed studies should be conducted to investigate the underlying reasons for the
detrimental effects on ileal morphology.

Moreover, dietary PP and FP did not alter the serum glutathione peroxidase, superoxide
dismutase, and catalase levels but decreased malondialdehyde (p < 0.05) in breast meat.

Revealed that investigated the effects of raw pomegranate pomace (5 and 10 g/kg) and
fermented pomegranate (5 and 10 g/kg) on performance, antioxidant activity, caecal
microbiota and ileal morphology in male broiler chickens. Compared to control diets.

Dietary PP and FP did not change the BW and FCR. Moreover, dietary PP and FP did not
alter the serum glutathione peroxidase, superoxide dismutase, and catalase levels but
decreased malondialdehyde (p < 0.05) in breast meat.

Caecal Clostridium perfringens count was decreased in broiler chickens of groups 10PP, 5FP
and 10FP.

However, PP and FP had detrimental effects on the ileum morphology of broiler chicks. The
VH was decreased in the 10PP, 5FP and 10FP groups compared with the control group (p <
0.01). CD was higher in the 5PP and 10FP groups than control and 10PP groups (p < 0.01).
The VH/CD ratio was also decreased in 5PP, 5FP, and 10FP groups (p < 0.01). These results
suggest that PP and FP have the potential to be used in broiler diets as antioxidant and
antimicrobial agents. However, detailed studies should be conducted to investigate the
underlying reasons for the detrimental effects on ileal morphology.

Pomegranate peel has antibacterial effects on various bacteria such as E. coli, S. aureus, and
Salmonella spp. thanks to its phenolic compounds (Gullon et al. 2016).
Ahmed and Yang (2017) indicated that PP can improve the intestinal microbiota of broiler
chickens due to high amounts of polyphenols, flavonoids and hydrolysable tannins, and by
acidifying the intestines by its low pH level (pH 3.56).

Sarica and Urkmez (2016) demonstrated that dietary supplementation of pomegranate peel
extract at 0.1 and 0.2 g/kg reduced total coliform and E. coli counts in the ileum and
enhanced the ileal Lactobacillus spp. count in broiler chicks. Similarly, caecal Lactobacillus
spp. count was raised by inclusion of pomegranate seed oil 20 g/kg diet in broilers (Rezvani
et al. 2018). Similar to the results on caecal C. perfringens count in the present study, Ahmed
and Yang (2017) reported that 10 g PP/kg diet diminished the E. coli counts in caecum and
ileum although no effect was observed on the E. coli counts at the inclusion of 5 g/kg in
broiler chickens. However, Rezvani et al. (2018) reported no changes in ileal E. coli and
Lactobacillus spp. counts of ileum and caecum by addition of pomegranate seed oil 20 g/kg
diet. Similarly, supplementation of PP 5 and 10 g /kg in broiler diets did not alter the
Lactobacillus spp. count in the ileum (Ahmed and Yang 2017).

Villus height, CD, VH:CD, and MMT are important indicators of intestinal development,
absorption capacity and health status (Gungor and Erener, 2020b).

Shorter VH, deeper CD and thinner muscularis mucosa may indicate the low absorption
capacity and also the presence of toxins (Xu et al. 2003). Ahmed et al. (2017) showed that the
lower levels of polyphenols, hydrolysable tannin and condensed tannin in PP have positive
effects on broilers, but higher levels exert a toxic effect on broiler chickens.

Some phenolic compounds at higher levels may cause a reduction in digestibility of nutrients
by binding to proteins in the intestinal lumen (Chamorro et al. 2013) and suppressing the
villus development (Viveros et al. 2011) in broiler chickens. The worsened intestinal
morphology may because of the detrimental effects of the condensed tannin and/or other
possible antinutritional compounds of PP. Indeed, the condensed tannin contents of PP (16.1
mg/kg PP) and FP (18.7 mg/kg FP) used in this study were higher than in previous studies by
Saleh et al. (2018) (6.72 mg/kg PP) and Hosseini-Vashan and Raei-Moghadam (2019) (1.3
mg/kg pomegranate pulp).

Nyamambi et al. (2007) demonstrated that high dietary condensed tannin caused detrimental
effects on intestinal morphology with reducing VH of broiler chickens. Similar to the result
of the present study, Sharifian et al. (2019) observed a tendency to impair the intestinal
morphology by raising CD of jejunum in broiler chicks with addition of PPE at 0.25–0.65
g/kg diet containing 53.8 mg condensed tannins per g.

Pomegranate peel has antibacterial effects on various bacteria such as E. coli, S. aureus, and
Salmonella spp. thanks to its phenolic compounds (Gullon et al. 2016).

Ahmed and Yang (2017) indicated that PP can improve the intestinal microbiota of broiler
chickens due to high amounts of polyphenols, flavonoids and hydrolysable tannins, and by
acidifying the intestines by its low pH level (pH 3.56).

Sarica and Urkmez (2016) demonstrated that dietary supplementation of PPE at 0.1 and 0.2
g/kg reduced total coliform and E. coli counts in the ileum and escalated the ileal
Lactobacillus spp. count in broiler chicks. Similarly, caecal Lactobacillus spp. count was
raised by inclusion of 20 g pomegranate seed oil per kg diet in broilers (Rezvani et al. 2018).

Similar to the results on caecal C. perfringens count in the present study,

Ahmed and Yang (2017) reported that 10 g PP/kg diet diminished the E. coli counts in
caecum and ileum although no effect was observed on the E. coli counts at the inclusion of 5
g/kg in broiler chickens.

However, Rezvani et al. (2018) reported no changes in ileal E. coli and Lactobacillus spp.
counts of ileum and caecum by addition of 20 g pomegranate seed oil per kg diet. Similarly,
supplementation of 5 and 10 g PP/kg in broiler diets did not alter the Lactobacillus spp. count
in the ileum (Ahmed and Yang 2017).